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Cell Signaling Technology Inc pim2 d1d2 rabbit mab
Pim2 D1d2 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+pim2/Pim-2+Rabbit+mAb/pm39747141__41467_2024_55572_MOESM2_ESM-61-0-5
Average 93 stars, based on 80 article reviews

pim2 d1d2 rabbit mab - by Bioz Stars, 2026-09

93/100 stars

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Article Title: Targeting PIM kinase as a therapeutic strategy in human hepatoblastoma
Article Snippet: Rabbit monoclonal anti-PIM1 (3247), anti-PIM2 (4730), anti-PIM3 (4165), anti-BAD (9239), and anti-phospho S112 BAD (5284) were from Cell Signaling Technology (Beverly, MA).

Article Title: EDC3 phosphorylation regulates growth and invasion through controlling P‐body formation and dynamics
Article Snippet: The following antibodies were purchased from Cell Signaling Technology: anti‐Pim1 (Cat#2907), anti‐Pim2 (Cat#4730), anti‐Pim3 (Cat#4165), anti‐AKT (Cat#9272), anti‐phospho‐AKT (S473, Cat#4058), anti‐HIS (Cat#2365), anti‐EDC3 (Cat #14495), anti‐DDX6 (Cat #9407), anti‐GFP (Cat#2555), anti‐phospho‐IRS1 (s1101, Cat#2385), anti‐eIF4B (Cat#3592), anti‐phospho‐eIF4B (Ser406, Cat#5399), anti‐GSK3B (Cat #9315), anti‐phospho‐GSK3B (Ser9, Cat #9336), anti‐S6 (Cat #9202), anti‐phospho‐S6 (Ser235/236, Cat#4856), and anti‐DCP1a (Cat#15365).

Article Title: Activation of Pim Kinases Is Sufficient to Promote Resistance to MET Small Molecule Inhibitors
Article Snippet: Antibodies and Reagents The following antibodies were purchased from Cell Signaling Technology: Anti-Pim-1 (Cat#3247), anti-Pim2 (Cat#4730), anti-Pim3 (Cat#4165) anti-MET (Cat#8198), anti-phospho-MET (Cat#3077), anti-BAD (Cat#9239), anti-phospho-BAD (Cat#5284), anti-eIF4B (Cat#3592), anti-phospho-eIF4B (S406, Cat#8151), anti-eIF4G (Cat#2498), anti-eIF4E(Cat#2067), anti-Myc-Tag(Cat#2272), anti-AKT (pan, Cat#4691), anti-phospho-AKT (S473, Cat#4058), anti-phospho-4E-BP1 (Cat#2855), anti-4E-BP1 (Cat#9452), anti-phospho-S6 (Cat#2215), anti-ERK (Cat#9102), anti-phospho-ERK (Cat#9101), anti-Bcl-2(Cat#4223), anti-Bim(Cat#2933), and anti-cleaved PARP(Cat#5625).

Article Title: EDC3 phosphorylation regulates growth and invasion through controlling P‐body formation and dynamics
Article Snippet: Antibodies and reagents The following antibodies were purchased from Cell Signaling Technology: anti‐Pim1 (Cat#2907), anti‐Pim2 (Cat#4730), anti‐Pim3 (Cat#4165), anti‐AKT (Cat#9272), anti‐phospho‐AKT (S473, Cat#4058), anti‐HIS (Cat#2365), anti‐EDC3 (Cat #14495), anti‐DDX6 (Cat #9407), anti‐GFP (Cat#2555), anti‐phospho‐IRS1 (s1101, Cat#2385), anti‐eIF4B (Cat#3592), anti‐phospho‐eIF4B (Ser406, Cat#5399), anti‐GSK3B (Cat #9315), anti‐phospho‐GSK3B (Ser9, Cat #9336), anti‐S6 (Cat #9202), anti‐phospho‐S6 (Ser235/236, Cat#4856), and anti‐DCP1a (Cat#15365).

Western Blot:

Article Title: PIM protein kinases regulate the level of the long noncoding RNA H19 to control stem cell gene transcription and modulate tumor growth
Article Snippet: The levels of PIM1 kinase (relative to ACTIN) and p‐IRS1 (S1101) proteins (relative to IRS1) were quantified and normalized to their respective control samples using imagej (NIH, https://imagej.nih.gov/ij ) software. .. Primary antibodies used for western blotting included anti‐PIM1 (Cell Signaling Technology, Cat # 2907), anti‐PIM2 (Cell Signaling Technology, Cat # 4730), anti‐PIM3 (Cell Signaling Technology, Cat # 4165), anti‐pIRS1‐S1101 (Cell Signaling Technology, Cat # S1101 Cat # 2385), anti‐insulin receptor substrate 1 (IRS1; Cell Signaling Technology, Cat # 06‐248), anti‐HA (Cell Signaling Technology, Cat # 14031), anti‐SOX2 [(E‐4) sc‐365823; Santa Cruz Biotech, Santa Cruz, CA, USA], anti‐OCT‐4 [(C‐10) sc‐5279; Santa Cruz Biotech], and anti‐NANOG [(5A10) sc‐134218; Santa Cruz Biotech]. .. HRP‐conjugated anti‐β‐actin (Cat # A3854) was purchased from Sigma‐Aldrich.

Blocking Assay:

Article Title: Inhibition of PIM2 in liver cancer decreases tumor cell proliferation in vitro and in vivo primarily through the modulation of cell cycle progression
Article Snippet: A volume equivalent to 20 μ g protein was loaded onto a 10% polyacrylamide gel, separated by SDS-PAGE, and transferred onto 0.45 μ m ImmobilonTM-P Transfer PVDF Membranes (Millipore). .. The membranes were blocked with a protein-free blocking buffer (Pierce), washed in TBST (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween-20), and incubated with primary antibodies diluted in blocking buffer: Anti-PIM2 (monoclonal, rabbit anti-human, 1:1,000, #4730, Cell Signaling Technology), anti-survivin (monoclonal, rabbit anti-human, 1:5,000, GTX62039, GeneTex), anti-PLK1 (monoclonal, rabbit anti-human, 1:1,000, #4513, Cell Signaling Technology), anti-vinculin (monoclonal, mouse anti-human, 1:5,000, V9131, Sigma-Aldrich) and anti-tubulin (monoclonal, mouse anti-human, 1:2,000, T5168, Sigma-Aldrich). .. The blots were incubated overnight at 4°C, washed in TBST and incubated for 1 h at room temperature with horseradish peroxidase-coupled goat anti-rabbit IgG (1:2,000; Cell Signaling Technology, #4414) or horseradish peroxidase-coupled goat anti-mouse IgG (1:5,000; Thermo Fisher Scientific, #31430) in blocking buffer.

Incubation:

Image Search Results

Estimated copies per cell of PIM1 and PIM2 protein from quantitative proteomics analysis of ( A ) OT1 CD8 T cells stimulated with SIINFEKL peptide for indicated times from published dataset or ( B ) naive ex vivo and 24 hr αCD3/αCD28 (TCR) activated WT CD8 T cells (see ( G, H )) for further details. ( C ) Fragments per kilobase million (FPKM) of Pim1 , Pim2, and Pim3 mRNA from published bulk RNAseq analysis of naive and 24 hr gp33-41 peptide stimulated P14 CD8 T cells. Lymph node cell suspensions from C57BL/6 (WT) and Pim1 KO/ Pim2 KO (Pim dKO) mice were activated for 24 hr with αCD3/αCD28 (both 0.5 µg/mL) and CD4 and CD8 T cell ( D ) FSC-A SSC-A profiles, ( E ) expression of surface activation markers (CD25, CD44, CD71) or CD8 T cell intracellular IFNγ were measured by flow cytometry. ( F ) Lymph node single-cell suspensions from WT and Pim dKO mice were labelled with CellTrace Violet (CTV), activated with αCD3/αCD28 (both 0.5 µg/mL) and CD4 and CD8 T cell CTV proliferation profiles were measured at indicated time points. ( G, H ) Lymph node cell suspensions from WT and Pim dKO mice were stimulated for 24 hr with αCD3/αCD28 (both 0.5 µg/mL) and activated CD4 and CD8 T cells were sorted for analysis by quantitative proteomics. Data was analysed using proteomic ruler method to estimate protein copy number per cell. An interactive version of the proteomics expression data is available for exploration on the Immunological Proteome Resource website: immpres.co.uk ( G ) Total protein content (µg/million cells) (one-way ANOVA), ( H ) Volcano plots of p-value (-log 10 ) versus fold-change (log 2 ) in protein copy number between Pim dKO and WT. Horizontal dotted line represents multi-test correction cut-off of q=0.05, vertical dotted line shows 1.5-fold change. Phosphoribosyl Pyrophosphate synthase 1 like 1 (Prps1l1), was found to be higher in Pim dKO CD8 T cells, but was a low confidence quantification (based on only two unique peptides) with no known function in T cells. Lymph node single-cell suspensions from WT and Pim dKO mice were labelled with CellTrace Violet (CTV) and ( I ) cells were cultured in IL-7 (5 ng/mL) +/- rapamycin (20 nM) and CD8 T cell numbers measured over time or ( J ) cells were activated with αCD3/αCD28 (both 0.5 µg/mL) +/- rapamycin (20 nM) and CD8 T cell mean division number was calculated over time (two-way ANOVA). Symbols in bar charts represent biological replicates, symbols in ( I ) represent the mean. Error bars show mean ± S.D. Flow cytometry dot plots and histograms are representative of ( D, E ) n=3, except for IFNγ staining which is n=2, ( F ) n=5, or show pooled data from ( I ) n=3–4 and ( J ) n=5 biological replicates, with data collected over at least two independent experiments. Quantitative proteomics was performed on biological triplicates. Figure 1—source data 1. Raw values plotted in .

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: Estimated copies per cell of PIM1 and PIM2 protein from quantitative proteomics analysis of ( A ) OT1 CD8 T cells stimulated with SIINFEKL peptide for indicated times from published dataset or ( B ) naive ex vivo and 24 hr αCD3/αCD28 (TCR) activated WT CD8 T cells (see ( G, H )) for further details. ( C ) Fragments per kilobase million (FPKM) of Pim1 , Pim2, and Pim3 mRNA from published bulk RNAseq analysis of naive and 24 hr gp33-41 peptide stimulated P14 CD8 T cells. Lymph node cell suspensions from C57BL/6 (WT) and Pim1 KO/ Pim2 KO (Pim dKO) mice were activated for 24 hr with αCD3/αCD28 (both 0.5 µg/mL) and CD4 and CD8 T cell ( D ) FSC-A SSC-A profiles, ( E ) expression of surface activation markers (CD25, CD44, CD71) or CD8 T cell intracellular IFNγ were measured by flow cytometry. ( F ) Lymph node single-cell suspensions from WT and Pim dKO mice were labelled with CellTrace Violet (CTV), activated with αCD3/αCD28 (both 0.5 µg/mL) and CD4 and CD8 T cell CTV proliferation profiles were measured at indicated time points. ( G, H ) Lymph node cell suspensions from WT and Pim dKO mice were stimulated for 24 hr with αCD3/αCD28 (both 0.5 µg/mL) and activated CD4 and CD8 T cells were sorted for analysis by quantitative proteomics. Data was analysed using proteomic ruler method to estimate protein copy number per cell. An interactive version of the proteomics expression data is available for exploration on the Immunological Proteome Resource website: immpres.co.uk ( G ) Total protein content (µg/million cells) (one-way ANOVA), ( H ) Volcano plots of p-value (-log 10 ) versus fold-change (log 2 ) in protein copy number between Pim dKO and WT. Horizontal dotted line represents multi-test correction cut-off of q=0.05, vertical dotted line shows 1.5-fold change. Phosphoribosyl Pyrophosphate synthase 1 like 1 (Prps1l1), was found to be higher in Pim dKO CD8 T cells, but was a low confidence quantification (based on only two unique peptides) with no known function in T cells. Lymph node single-cell suspensions from WT and Pim dKO mice were labelled with CellTrace Violet (CTV) and ( I ) cells were cultured in IL-7 (5 ng/mL) +/- rapamycin (20 nM) and CD8 T cell numbers measured over time or ( J ) cells were activated with αCD3/αCD28 (both 0.5 µg/mL) +/- rapamycin (20 nM) and CD8 T cell mean division number was calculated over time (two-way ANOVA). Symbols in bar charts represent biological replicates, symbols in ( I ) represent the mean. Error bars show mean ± S.D. Flow cytometry dot plots and histograms are representative of ( D, E ) n=3, except for IFNγ staining which is n=2, ( F ) n=5, or show pooled data from ( I ) n=3–4 and ( J ) n=5 biological replicates, with data collected over at least two independent experiments. Quantitative proteomics was performed on biological triplicates. Figure 1—source data 1. Raw values plotted in .

Article Snippet: Blots were probed with the following primary antibodies: S6K (5G10, Cell Signaling Technology, cat# 2217 S), S6K p-T389 (108D2, Cell Signaling Technology, cat# 9234 S), STAT5 p-Y694 (C11C5, Cell Signalling Technology, cat# 9359 S), Pim1 (12H8, Santa Cruz, cat# SC-13513), Pim2 (1D12, Santa Cruz, cat# SC-13514).

Techniques: Quantitative Proteomics, Ex Vivo, Expressing, Activation Assay, Flow Cytometry, Cell Culture, Staining

Pim1 KO mice have the Pim1 gene deleted from 98 codons post-ATG start site onwards (mid exon 4) and ablation of kinase activity was confirmed in . In Pim2 KO mice exons 1–3 are deleted from the Pim2 gene . Summed peptide intensities from unnormalized proteomics data collected as described in from ( A ) before the PIM1 deletion point (left) and after the deletion point (right) and ( B ) whole of PIM2 protein confirms deletion of functional PIM1 and PIM2 protein. Figure 1—figure supplement 2—source data 1. Raw values plotted in .

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: Pim1 KO mice have the Pim1 gene deleted from 98 codons post-ATG start site onwards (mid exon 4) and ablation of kinase activity was confirmed in . In Pim2 KO mice exons 1–3 are deleted from the Pim2 gene . Summed peptide intensities from unnormalized proteomics data collected as described in from ( A ) before the PIM1 deletion point (left) and after the deletion point (right) and ( B ) whole of PIM2 protein confirms deletion of functional PIM1 and PIM2 protein. Figure 1—figure supplement 2—source data 1. Raw values plotted in .

Techniques: Activity Assay, Functional Assay

( A ) OT1 lymph node cell suspensions were SIINFEKL peptide activated for 36 hr, washed then cultured with no cytokine, IL-15 (20 ng/mL) or IL-2 (20 ng/mL) for 4 or 24 hr. Western blots of PIM1 (two isoforms of 44 and 34 kDa, non-specific band indicated by *), PIM2 (three isoforms of 40, 37, and 34 kDa) or pSTAT5 Y694 expression. ( B ) Schematic of cytokine driven memory and effector CD8 T cell expansion and differentiation cultures. Lymph node or spleen cell suspensions were activated for 2 days with TCR stimulus + cytokine, washed, then split daily into fresh media + cytokine. ( C ) WT (Ly5.1) and Pim dKO LN suspensions were mixed at a 50:50 ratio for T cells and cultured as outlined in ( B ) with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), and CD8 T cell number was measured daily. ( D ) WT and Pim dKO T cells were expanded with IL-15 in separate cultures as per ( B, C ) and % live cells (PI-ve) were assessed on days 4 and 6 (two-way ANOVA). ( E–J ) WT and Pim dKO CD8 T cells were activated with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), expanded with IL-15 as per ( B ), with an additional CD4 T cell magnetic depletion step on day 3 of culture. CD8 T cells were harvested on day 6 for parallel RNAseq and proteomic analysis. An interactive version of the proteomics expression data is available for exploration on the Immunological Proteome Resource website: immpres.co.uk ( E ) Fold-change in mRNA expression between Pim dKO and WT versus average mRNA expression (TPM). mRNA expression (Transcripts per million, TPM) of ( F ) secondary lymphoid homing receptors Sell, Ccr7, S1pr1 and ( G ) key transcription factors involved in CD8 T cell memory differentiation and maintenance Tcf7, Klf2, Foxo1, Foxo3, Id3. ( H ) WT vs Pim dKO protein copy numbers, differentially expression proteins (FC >1.5, q<0.05) are highlighted in red ( I ) Protein copy numbers per cell for key mitochondrial proteins DRP1, OPA1, and CPT1A. ( J ) WT vs Pim dKO protein copy numbers, mitochondrial proteins (as defined in MitoCarta 3.0) are highlight in pink. Symbols in bar charts represent biological replicates, symbols in ( C, E, H, J ) represent the mean. Error bars show mean ± S.D. Data are representative of ( A ) n=3 or show pooled data from ( C ) n=4, and ( D ) n=5 biological replicates with data collected over at least two independent experiments. Quantitative proteomics and RNAseq was performed on biological triplicates. ** q≤0.01, fold-change (FC) shown on bar graphs when q<0.05. Figure 2—source data 1. PDF files containing labelled and uncropped images for western blots displayed in . Figure 2—source data 2. Original files for western blot images displayed in . Figure 2—source data 3. Raw values plotted in .

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: ( A ) OT1 lymph node cell suspensions were SIINFEKL peptide activated for 36 hr, washed then cultured with no cytokine, IL-15 (20 ng/mL) or IL-2 (20 ng/mL) for 4 or 24 hr. Western blots of PIM1 (two isoforms of 44 and 34 kDa, non-specific band indicated by *), PIM2 (three isoforms of 40, 37, and 34 kDa) or pSTAT5 Y694 expression. ( B ) Schematic of cytokine driven memory and effector CD8 T cell expansion and differentiation cultures. Lymph node or spleen cell suspensions were activated for 2 days with TCR stimulus + cytokine, washed, then split daily into fresh media + cytokine. ( C ) WT (Ly5.1) and Pim dKO LN suspensions were mixed at a 50:50 ratio for T cells and cultured as outlined in ( B ) with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), and CD8 T cell number was measured daily. ( D ) WT and Pim dKO T cells were expanded with IL-15 in separate cultures as per ( B, C ) and % live cells (PI-ve) were assessed on days 4 and 6 (two-way ANOVA). ( E–J ) WT and Pim dKO CD8 T cells were activated with TCR stimulus αCD3/αCD28 (both 0.5 µg/mL) + cytokine IL-15 (20 ng/mL), expanded with IL-15 as per ( B ), with an additional CD4 T cell magnetic depletion step on day 3 of culture. CD8 T cells were harvested on day 6 for parallel RNAseq and proteomic analysis. An interactive version of the proteomics expression data is available for exploration on the Immunological Proteome Resource website: immpres.co.uk ( E ) Fold-change in mRNA expression between Pim dKO and WT versus average mRNA expression (TPM). mRNA expression (Transcripts per million, TPM) of ( F ) secondary lymphoid homing receptors Sell, Ccr7, S1pr1 and ( G ) key transcription factors involved in CD8 T cell memory differentiation and maintenance Tcf7, Klf2, Foxo1, Foxo3, Id3. ( H ) WT vs Pim dKO protein copy numbers, differentially expression proteins (FC >1.5, q<0.05) are highlighted in red ( I ) Protein copy numbers per cell for key mitochondrial proteins DRP1, OPA1, and CPT1A. ( J ) WT vs Pim dKO protein copy numbers, mitochondrial proteins (as defined in MitoCarta 3.0) are highlight in pink. Symbols in bar charts represent biological replicates, symbols in ( C, E, H, J ) represent the mean. Error bars show mean ± S.D. Data are representative of ( A ) n=3 or show pooled data from ( C ) n=4, and ( D ) n=5 biological replicates with data collected over at least two independent experiments. Quantitative proteomics and RNAseq was performed on biological triplicates. ** q≤0.01, fold-change (FC) shown on bar graphs when q<0.05. Figure 2—source data 1. PDF files containing labelled and uncropped images for western blots displayed in . Figure 2—source data 2. Original files for western blot images displayed in . Figure 2—source data 3. Raw values plotted in .

Techniques: Cell Culture, Western Blot, Expressing, Quantitative Proteomics

( A ) Estimated copies per cell of PIM1 and PIM2 protein from published quantitative proteomics analysis ; of CD8 T cells expanded in IL-2 or IL-15 as outlined in . ( B–D, F, G ) WT (Ly5.1) and Pim dKO lymph node or spleen single-cell suspensions were mixed at a 50:50 ratio of T cells, activated for 2 days with αCD3/αCD28 (both 0.5 µg/mL) and IL-2 (20 ng/mL), washed then split into fresh medium containing IL-2 (20 ng/mL) daily (as per ). Some of the mixed cell suspensions were also cultured in IL-7 (5 ng/mL) to sustain a naive T cell reference. ( B ) WT and Pim dKO CTL were treated 1 hr +/- Jak1/3 inhibitor Tofacitinib (100 nM; negative control) before pSTAT5 Y694 expression was measured on day 3 and 6 of culture, ( C ) surface CD25 expression was measured on days 3 and 6 of culture, ( D ) CD8 T cell number vs time was calculated, ( F ) CD8 T cell FSC-A, SSC-A and surface activation markers (CD44, CD71) were measured on days 3 and 6 of culture ( G ) expression of adhesion molecule CD62L was measured daily. ( E ) WT and Pim dKO T cells were activated and expanded with IL-2 as per ( B-D ) and ( F, G ) except in separate cultures and % live cells (PI-ve) was assessed on days 4 and 6 (two-way ANOVA). Symbols in bar charts represent biological replicates, symbols in ( D ) represent the mean. Error bars show mean ± S.D. Data are representative of ( B, G ) n=4, ( C, F ) n=6 or show pooled data from ( D ) n=4, ( E ) n=6 biological replicates with data collected over at least two independent experiments. Figure 3—source data 1. Raw values plotted in .

Journal: eLife

Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking

doi: 10.7554/eLife.98622

Figure Lengend Snippet: ( A ) Estimated copies per cell of PIM1 and PIM2 protein from published quantitative proteomics analysis ; of CD8 T cells expanded in IL-2 or IL-15 as outlined in . ( B–D, F, G ) WT (Ly5.1) and Pim dKO lymph node or spleen single-cell suspensions were mixed at a 50:50 ratio of T cells, activated for 2 days with αCD3/αCD28 (both 0.5 µg/mL) and IL-2 (20 ng/mL), washed then split into fresh medium containing IL-2 (20 ng/mL) daily (as per ). Some of the mixed cell suspensions were also cultured in IL-7 (5 ng/mL) to sustain a naive T cell reference. ( B ) WT and Pim dKO CTL were treated 1 hr +/- Jak1/3 inhibitor Tofacitinib (100 nM; negative control) before pSTAT5 Y694 expression was measured on day 3 and 6 of culture, ( C ) surface CD25 expression was measured on days 3 and 6 of culture, ( D ) CD8 T cell number vs time was calculated, ( F ) CD8 T cell FSC-A, SSC-A and surface activation markers (CD44, CD71) were measured on days 3 and 6 of culture ( G ) expression of adhesion molecule CD62L was measured daily. ( E ) WT and Pim dKO T cells were activated and expanded with IL-2 as per ( B-D ) and ( F, G ) except in separate cultures and % live cells (PI-ve) was assessed on days 4 and 6 (two-way ANOVA). Symbols in bar charts represent biological replicates, symbols in ( D ) represent the mean. Error bars show mean ± S.D. Data are representative of ( B, G ) n=4, ( C, F ) n=6 or show pooled data from ( D ) n=4, ( E ) n=6 biological replicates with data collected over at least two independent experiments. Figure 3—source data 1. Raw values plotted in .

Techniques: Quantitative Proteomics, Cell Culture, Negative Control, Expressing, Activation Assay

Fig. 1 JAK2 ATP-competitive inhibitors treatment leads to paradox JAK2 hyperphosphorylation. Ba/F3 cells expressing the JAK2-V617F and JAK2-WT were treated with the indicated concentration of the ruxolitinib (A) fedratinib (B) and lestaurtinib (C) for 3 h, and lysates were prepared and subjected to western blotting for the pJAK2, JAK2, pSTAT5 and STAT5. Ba/F3 cells expressing the JAK1-V658F and JAK1-WT were treated for 3.5 h with ruxolitinib, and lysates were analyzed for activation of JAK1 and STAT3 (D). Ba/F3 cells stably expressing the CSF3R-T618I and CSF3R-WT treated with indicated concentrations of ruxolitinib (E) and G-CSF (F) for 3 h, and lysates were subjected to western blotting for the pTYK2, TYK2, pSTAT3, STAT3, pSTAT5 and STAT5. Ba/F3 cells expressing the JAK3-WT were treated with indicated concentrations of ruxolitinib in the presence of interleukin-2 (IL-2), and lysates were subjected to western blotting for the pJAK3, JAK3, pSTAT3 and STAT3 (G).

Journal: Leukemia

Article Title: Ruxolitinib mediated paradoxical JAK2 hyperphosphorylation is due to the protection of activation loop tyrosines from phosphatases.

doi: 10.1038/s41375-025-02594-7

Figure Lengend Snippet: Fig. 1 JAK2 ATP-competitive inhibitors treatment leads to paradox JAK2 hyperphosphorylation. Ba/F3 cells expressing the JAK2-V617F and JAK2-WT were treated with the indicated concentration of the ruxolitinib (A) fedratinib (B) and lestaurtinib (C) for 3 h, and lysates were prepared and subjected to western blotting for the pJAK2, JAK2, pSTAT5 and STAT5. Ba/F3 cells expressing the JAK1-V658F and JAK1-WT were treated for 3.5 h with ruxolitinib, and lysates were analyzed for activation of JAK1 and STAT3 (D). Ba/F3 cells stably expressing the CSF3R-T618I and CSF3R-WT treated with indicated concentrations of ruxolitinib (E) and G-CSF (F) for 3 h, and lysates were subjected to western blotting for the pTYK2, TYK2, pSTAT3, STAT3, pSTAT5 and STAT5. Ba/F3 cells expressing the JAK3-WT were treated with indicated concentrations of ruxolitinib in the presence of interleukin-2 (IL-2), and lysates were subjected to western blotting for the pJAK3, JAK3, pSTAT3 and STAT3 (G).

Article Snippet: STAT5 (G-2), ID1, PIM1, PIM2 pJAK2 (21870-R) antibodies were obtained from Santacruz Biotechnology (Heidelberg, Germany).

Techniques: Expressing, Concentration Assay, Western Blot, Activation Assay, Stable Transfection

Figure 4 The effect of MXJDH on macrophage polarization and Pim2/NF-κB pathway. (A) Flow cytometry analysis of the effect of MXJDH on CD86 and CD206 expression in LPS-treated macrophages. (B) Co-IP analysis of the formation of Pim2-p65. (C and D) Effect of MXJDH and Dexamethasone on inflammatory cytokines TNF-α and IL-10 in LPS-treated macrophages (n=3). (E) Protein expression level of p65, Pim2, IL-6, IL-10 and TNF-α. (F–J) Statistical analysis of p65, Pim2, IL-6, IL-10 and TNF-α protein levels (n=3). Data were shown as mean ± SD and were analyzed using one-way ANOVA (nonparametric) analysis. *P < 0.05, **p <0.01, ***P < 0.001, VS model group.

Journal: Journal of Inflammation Research

Article Title: Modified Xi-Jiao-Di-Huang Decoction Alleviates Sepsis via Regulating Macrophage Polarization by Inhibiting the PIM2/NF-κB Pathway

doi: 10.2147/jir.s509734

Figure Lengend Snippet: Figure 4 The effect of MXJDH on macrophage polarization and Pim2/NF-κB pathway. (A) Flow cytometry analysis of the effect of MXJDH on CD86 and CD206 expression in LPS-treated macrophages. (B) Co-IP analysis of the formation of Pim2-p65. (C and D) Effect of MXJDH and Dexamethasone on inflammatory cytokines TNF-α and IL-10 in LPS-treated macrophages (n=3). (E) Protein expression level of p65, Pim2, IL-6, IL-10 and TNF-α. (F–J) Statistical analysis of p65, Pim2, IL-6, IL-10 and TNF-α protein levels (n=3). Data were shown as mean ± SD and were analyzed using one-way ANOVA (nonparametric) analysis. *P < 0.05, **p <0.01, ***P < 0.001, VS model group.

Article Snippet: Reagents NF-κB-p65 Antibody (Cat No. 21014), NF-κB p65 Mouse Monoclonal Antibody (Cat No. 38054), PIM2 Rabbit mAb (Cat No. 56914), IL10 Antibody (Cat No. 38392), and goat anti-rabbit IgG Secondary Antibody (Cat No. L3012) were purchased from Signalway Antibodies (Jiangsu, China).

Techniques: Flow Cytometry, Expressing, Co-Immunoprecipitation Assay

Figure 5 MXJDH ameliorates sepsis by inhibiting the Pim2/NF-κB pathway. (A–E) Statistical analysis of mRNA expression levels of p65, Pim2, IL-6, IL-10 and TNF-α (n=3). (F–K) Protein expression level of p65, Pim2, IL-6, IL-10 and TNF-α and statistical analysis (n=3). (L) Protein expression levels of p65 and Pim2 in cytoplasm and nucleus. (M) Representative images of P65, Pim2 immunofluorescence staining, and nuclear staining by DAPI. Scale bars, 20 µm as indicated. Data were shown as mean ± SD and were analyzed using one-way ANOVA (nonparametric) analysis. *P < 0.05, **p <0.01, ***P < 0.001, ****P < 0.0001, VS model group.

Journal: Journal of Inflammation Research

Article Title: Modified Xi-Jiao-Di-Huang Decoction Alleviates Sepsis via Regulating Macrophage Polarization by Inhibiting the PIM2/NF-κB Pathway

doi: 10.2147/jir.s509734

Figure Lengend Snippet: Figure 5 MXJDH ameliorates sepsis by inhibiting the Pim2/NF-κB pathway. (A–E) Statistical analysis of mRNA expression levels of p65, Pim2, IL-6, IL-10 and TNF-α (n=3). (F–K) Protein expression level of p65, Pim2, IL-6, IL-10 and TNF-α and statistical analysis (n=3). (L) Protein expression levels of p65 and Pim2 in cytoplasm and nucleus. (M) Representative images of P65, Pim2 immunofluorescence staining, and nuclear staining by DAPI. Scale bars, 20 µm as indicated. Data were shown as mean ± SD and were analyzed using one-way ANOVA (nonparametric) analysis. *P < 0.05, **p <0.01, ***P < 0.001, ****P < 0.0001, VS model group.

Techniques: Expressing, Immunofluorescence, Staining

Figure 6 The effect of MXJDH on macrophages in sepsis mice. (A) Representative images of F4/80, Pim2 immunofluorescence staining, and nuclear staining by DAPI. Scale bars, 25 µm as indicated. (B) Representative images of CD86, CD206 immunofluorescence staining, and nuclear staining by DAPI. Scale bars, 100 µm as indicated.

Journal: Journal of Inflammation Research

Article Title: Modified Xi-Jiao-Di-Huang Decoction Alleviates Sepsis via Regulating Macrophage Polarization by Inhibiting the PIM2/NF-κB Pathway

doi: 10.2147/jir.s509734

Figure Lengend Snippet: Figure 6 The effect of MXJDH on macrophages in sepsis mice. (A) Representative images of F4/80, Pim2 immunofluorescence staining, and nuclear staining by DAPI. Scale bars, 25 µm as indicated. (B) Representative images of CD86, CD206 immunofluorescence staining, and nuclear staining by DAPI. Scale bars, 100 µm as indicated.

Techniques: Immunofluorescence, Staining

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